The following description of the background of the invention is provided to aid in understanding the claimed invention, but it is not admitted to constitute or describe prior art to the claimed invention and should in no way be construed as limiting the claimed invention.
The traditional alkaline lysis process for isolation of plasmid DNA from bacterial cells is described in Birnboim, H. C. and J. Doly, Nucleic Acids Res. 7:1513, 1979 and is also described in J. F. Burke, Nucleic Acids Res. 8:2989, 1981. It makes use of several laboratory-scale apparatuses and manual operations which are not suitable for large-scale manufacturing. In addition, the traditional alkaline lysis process is not suitable for making pharmaceutical grade material for human use, since inconsistent process performance may result in an unacceptable level of sheared host chromosomal DNA in the final plasmid product. The difficulty of theoretical study of some of the critical parameters for manual operations, like shear force, also make the traditional process unlikely to become a large scale manufacturing process.
Such traditional processes consist of three stages. The first stage is cell resuspension, which normally utilizes manual stirring or a magnetic stirrer, and a homogenizer or impeller mixer to resuspend cells in the resuspension buffer. Manual stirring and magnetic stirring are only appropriate for laboratory scale preparations.
The second stage is cell lysis. It is desirable to minimize shear of host chromosomal DNA at this stage, because it is difficult to remove sheared chromosomal DNA in the downstream purification process (due to the similarity in properties between sheared chromosomal DNA and plasmid DNA). The lysis is normally carried out by manual swirling or magnetic stirring in order to mix the resuspended cells with lysis solution (consisting of diluted alkali (base) and detergents); then holding the mixture at room temperature (20–25 degrees Celsius) or on ice for a period of time, such as 5 minutes, to complete lysis. As noted above, manual swirling and magnetic stirring are not scalable. In addition, it is difficult to optimize the process and obtain consistent process performance due to the number of operation parameters, including operator to operator variability.
The third stage is neutralization and precipitation of host contaminants. Lysate from the second stage is normally mixed with a cold neutralization solution by gentle swirling or magnetic stirring to acidify the lysate before setting in ice for 10–30 minutes to facilitate the denaturation and precipitation of high molecular weight chromosomal DNA, host proteins, and other host molecules. Again, both manual swirling and magnetic, stirring are not scalable. An ice bath is not convenient if a long holding time is desired in a large scale process, because a large quantity of water must be removed and a large quantity of ice/or ice-NaCl mixture needs to be added periodically to maintain a steady temperature.
Once the plasmid DNA is extracted from the lysed cells its purification has become a routine and important procedure for the molecular biologist. However, the scale for these purifications, often referred to as “mini-preps”, is usually less than about 1 milligram of plasmid DNA. These small scale preps isolate plasmid DNA from the supernatant of lysed bacterial cells using a variety of techniques, such as ethanol precipitation. For slightly larger scale preparations, the primary techniques employed use cesium chloride centrifugation, binding and eluting to silica resins (in the presence of chaotropic salts) or binding and eluting with various anionic chromatography resins. In addition, other techniques are sometimes used in combination with the resins mentioned, e.g., PEG and/or alcohol precipitation, RNase treatment, and phenol/chloroform extraction. There are also some plasmid purifications performed using analytical HPLC, in particular reverse phase HPLC to separate different plasmid forms using organic solvent systems.
Reverse phase chromatography (“RPC”) is generally practiced by binding compounds of interest to a chromatography support in an aqueous solution and eluting with increasing amounts of an organic solvent, such as acetonitrile or alcohol. This approach has been used by a number of investigators to separate DNA, especially oligonucleotides and small (less than 1,000 base pair) restriction fragments. It has also been used to separate open circular and supercoiled plasmids. However, RPC solvents present volatility, fire, health and waste disposal risks. In addition, RPC of nucleic acids frequently requires the use of an ion-pairing agent (e.g., triethylammonium acetate or TEAA) which can be difficult to remove from the DNA and can be toxic to cultured cells.
The “mini-prep” procedures described above were designed for purifying small amounts of plasmid DNA and in general they have not been suitable for large-scale, high throughput purification processes. Large scale purification of plasmid DNA may magnify the contaminants in the final purifications in the final preparations, which usually go undetected in mini-preps. Anion-exchange chromatography as a single chromatography step is unlikely to remove enough of the contaminants which would be necessary for a therapeutic product. Thus, additional processes should probably be included to increase the purity of the plasmid DNA. In addition, it would be useful to have a scalable process which could also resolve various plasmid forms.
Two contaminants which may be particularly troublesome are RNA and chromosomal DNA. Many mini-prep procedures attempt to remove RNA using one or several RNase enzymes which degrade the RNA to ribonucleotides and small oligoribonucleotides. These can then be separated from the plasmid DNA using any of a variety of techniques, including alcohol precipitation, size exclusion chromatography, anion exchange chromatography, etc. However, the use of RNase is undesirable in large scale (equal to or greater than 50–100 mg of plasmid) purification. RNase is an expensive material that is generally not reused. Large scale RNase reactions can be difficult to perform in batch mode with appropriate control of time, temperature and other reaction conditions. Also, RNase is typically isolated from bovine pancreas. As such, it is a possible source of mammalian pathogens, especially retrovirus and bovine spongiform encephalopathy (BSE). Use of such materials in making plasmids for human use presents significant safety and regulatory issues.
Another approach to removal of RNA involves differential precipitations whereby plasmid DNA is precipitated while RNA remains in solution or vice versa. An example is described in WO 95/21250 in which polyethylene glycol (PEG) is used to precipitate RNA from a solution containing both RNA and plasmid. Similar techniques have been described. A disadvantage of these techniques as typically practiced is that the plasmid is first partially purified from the lysate by removing solid debris, precipitated proteins and other solids, and optionally by alcohol precipitation of the nucleic acids (including RNA and plasmid). Differential precipitation is then applied to the partially purified mixture of RNA and plasmid. This approach to differential precipitation has required multiple steps, increasing the time effort and complexity of the process, and introducing more opportunities for loss of plasmid.
Removing chromosomal DNA derived from the bacterial host is also a challenging task in plasmid purification. In a typical “mini-prep”, chromosomal DNA is removed primarily during the lysis and neutralization steps. Large fragments of chromosomal DNA are bound to proteins and membrane fragments, and are carried into the precipitate during the neutralization step. However, it is well known that if the chromosomal DNA is sheared to smaller size (≦ about 10 kb), it is not efficiently precipitated and contaminates the plasmid DNA. Thus, it has been assumed that during mini preps, one must avoid vigorous mixing and shearing of the lysate. This is difficult to achieve at larger scales due to the known difficulties of mixing large volumes of liquid. Thus, it is desirable to have a way to separate plasmid DNA from chromosomal DNA subsequent to lysis and neutralization.
One approach to this is CsCl/ethidium bromide density gradients. These are very effective for small amounts of plasmid (≦ 1 mg) that are not intended for human use. They are not generally suitable for scaling up to over 100 mg lots due to the high cost of necessary equipment. They are also not generally suitable for producing plasmid for human use because ethidium bromide binds tightly to DNA, is difficult to remove quantitatively, and is a known mutagen and suspected carcinogen.
Thus, there are several clear needs for large scale plasmid purification. These include a method to mix large volumes of lysate with low shear, a method to precipitate RNA directly from a lysate without prior additional purification, and a method to separate sheared chromosomal DNA from plasmid DNA.
Other methods for purification of larger amounts of plasmid DNA are not ideal and leave significant room for improvement. For example, the method described in WO 95/21250, published August 10, 1995 involves multiple precipitations, plasmid precipitations, low capacity size exclusions, and requires flammable alcohols. Similarly, the method described in WO 96/02658, published Feb. 1, 1996, requires lysozyme, may require RNase, and requires flammable alcohols.